Asif Naeem

Metagenomic Analyses of Viruses in Stool Samples from Children with Acute Flaccid Paralysis

ABSTRACT We analyzed viral nucleic acids in stool samples collected from 35 South Asian children with nonpolio acute flaccid paralysis (AFP). Sequence-independent reverse transcription and PCR amplification of capsid-protected, nuclease-resistant viral nucleic acids were followed by DNA sequencing and sequence similarity searches. Limited Sanger sequencing (35 to 240 subclones per sample) identified an average of 1.4 distinct eukaryotic viruses per sample, while pyrosequencing yielded 2.6 viruses per sample. In addition to bacteriophage and plant viruses, we detected known enteric viruses, including rotavirus, adenovirus, picobirnavirus, and human enterovirus species A (HEV-A) to HEV-C, as well as numerous other members of the Picornaviridae family, including parechovirus, Aichi virus, rhinovirus, and human cardiovirus. The viruses with the most divergent sequences relative to those of previously reported viruses included members of a novel Picornaviridae genus and four new viral species (members of the Dicistroviridae, Nodaviridae, and Circoviridae families and the Bocavirus genus). Samples from six healthy contacts of AFP patients were similarly analyzed and also contained numerous viruses, particularly HEV-C, including a potentially novel Enterovirus genotype. Determining the prevalences and pathogenicities of the novel genotypes, species, genera, and potential new viral families identified in this study in different demographic groups will require further studies with different demographic and patient groups, now facilitated by knowledge of these viral genomes.

A highly prevalent and genetically diversified Picornaviridae genus in South Asian children

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Cardioviruses Are Genetically Diverse and Cause Common Enteric Infections in South Asian Children

ABSTRACT Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The Cardiovirus genus within the Picornaviridae family is currently made up of two viral species, Theilovirus and Encephalomyocarditis virus. Until recently, only a single strain of cardioviruses (Vilyuisk virus within the Theilovirus species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus (Saffold virus [SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.

Multiple Independent Emergences of Type 2 Vaccine-Derived Polioviruses during a Large Outbreak in Northern Nigeria

ABSTRACT Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5′-untranslated region [5′-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5′-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

Genomic Characterization of Novel Human Parechovirus Type

Using a simple metagenomic approach, we identified a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.

Molecular epidemiology of Hepatitis B virus genotypes in Pakistan

BackgroundEight genotypes of Hepatitis B virus designated A-H, have been known but in Pakistan, no such data is available on the prevalent HBV genotypes. Therefore, the subject study was conducted to determine HBV genotypes in the indigenous Pakistani population.MethodsA total of 690 individuals were enrolled for HBV screening with EIA and nested PCR. Positive samples were further analyzed to determine HBV genotypes (A-F) by multiplex-PCR using type specific primers.Results110 (15.94%) individuals were positive for HBV, including 64% males and 36% females. Out of these, 66 samples (65.34%) were classified into genotype D, 27 (26.73%) were of genotype B while 5(4.95%) had genotype A. In 3 (2.98%) samples, multiple genotypes were detected (genotype A+B; 2(1.99%) and genotypes B+D; 1(0.99%). Nine (8.18%) samples remained untyable.ConclusionIn Asia, genotypes B and C are the most prevalent but our study reveals that genotype D is predominant and HBV infection constitutes a significant health problem in Pakistan.

Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan

BackgroundEnteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder.MethodsStool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases.ResultsNPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers.ConclusionThe present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.

Common genotypes of Hepatitis B virus prevalent in injecting drug abusers (addicts) of North West Frontier Province of Pakistan.

BackgroundThe epidemiological significance of Hepatitis B virus genotypes has been well established and becoming an essential concern day by day however, much little is known about the mixed infection with more than one Hepatitis B virus genotypes and their clinical relevance.MethodsIntravenous drug abusers are considered as a major risk group for the acquisition and transmission of blood borne infections like hepatitis B, however, in Pakistan, no such data has ever been reported about the epidemiology of HBV and its genotypes in Injecting Drug Users. 250 individuals were analyzed for hepatitis B virus genotypes after prior screening with serological assay for the detection of HBsAg.Results56 (22.4%) individuals were found positive on ELSIA for HBsAg. The genotype distribution was found to be as: genotype D, 62.5%; genotype A, 8.92% while 28.57% individuals were found to be infected with a mixture of genotype A and D.ConclusionThere is an urgent need of the time to develop public health care policies with special emphasis towards the control of HBV transmission through high risk groups especially Injecting Drug Users.

Genetic characterization of rotavirus subtypes in Pakistan-first report of G12 genotype from Pakistan under WHO-Eastern Mediterranean region.

Rotaviruses are among the major causes of gastroenteritis and diarrhea among children in developed as well as the developing countries. The rapidly evolving strain prevalence and circulation have resulted in the emergence of novel strains over the period worldwide. The introduction of G12 prototype in 1987 from Philippines and subsequently re-emergence among most of the Asian countries along with USA and Europe has provoked new research horizons to address the global distribution of rotavirus serotypes. These newly emerging subtypes and their sustenance among the population have posed tremendous challenge to the development of an effectual vaccine with heterotypic protective efficacy. In Pakistan, no data is available regarding the prevalent rotavirus serotypes; therefore, this is the first study to report the prevalence of G12 strain in Pakistan in hospitalized children with diarrhea addressing a dire need of further large-scale epidemiological surveys to resolve the underlying rotavirus isolates in both the hospitalized and the community neonatal and child population before formulating the vaccine introduction policies in the countrys routine immunization program.

Neuropathogenicity of Two Saffold Virus Type 3 Isolates in Mouse Models

Objective Saffold virus (SAFV), a picornavirus, is occasionally detected in children with acute flaccid paralysis, meningitis, and cerebellitis; however, the neuropathogenicity of SAFV remains undetermined. Methods The virulence of two clinical isolates of SAFV type 3 (SAFV-3) obtained from a patient with aseptic meningitis (AM strain) and acute upper respiratory inflammation (UR strain) was analyzed in neonatal and young mice utilizing virological, pathological, and immunological methods. Results The polyproteins of the strains differed in eight amino acids. Both clinical isolates were infective, exhibited neurotropism, and were mildly neurovirulent in neonatal ddY mice. Both strains pathologically infected neural progenitor cells and glial cells, but not large neurons, with the UR strain also infecting epithelial cells. UR infection resulted in longer inflammation in the brain and spinal cord because of demyelination, while the AM strain showed more infectivity in the cerebellum in neonatal ddY mice. Additionally, young BALB/c mice seroconverted following mucosal inoculation with the UR, but not the AM, strain. Conclusions Both SAFV-3 isolates had neurotropism and mild neurovirulence but showed different cell tropisms in both neonatal and young mouse models. This animal model has the potential to recapitulate the potential neuropathogenicity of SAFV-3.